Mycotoxin

Mycotoxins likely have existed for as long as crops have been grown but recognition of the true chemical nature of such entities of fungal metabolism was not known until recent times. Conjecturally, there is historical evidence of their presence back as far as the time reported in the Dead Sea Scrolls. Evidence of their periodic, historical occurrence exists until the recognition of aflatoxins in the early 1960s. At that time mycotoxins were considered as a storage phenomenon whereby grains becoming moldy during storage allowed for the production of these secondary metabolites proven to be toxic when consumed by man and other animals. Subsequently, aflatoxins and mycotoxins of several kinds were found to be formed during development of crop plants in the field. The determination of which of the many known mycotoxins are significant can be based upon their frequency of occurrence and/or the severity of the disease that they produce, especially if they are known to be carcinogenic. Among the mycotoxins fitting into this major group would be the aflatoxins, deoxynivalenol, fumonisins, Zearalenone, T-2 toxin, ochratoxin and certain ergot alkaloids. The diseases (mycotoxicoses) caused by these mycotoxins are quite varied and involve a wide range of susceptible animal species including humans. Most of these diseases occur after consumption of mycotoxin contaminated grain or products made from such grains but other routes of exposure exist. The diagnosis of mycotoxicoses may prove to be difficult because of the similarity of signs of disease to those caused by other agents. Therefore, diagnosis of a mycotoxicoses is dependent upon adequate testing for mycotoxins involving sampling, sample preparation and analysis [2].

Zearalenone (ZEA) is a mycotoxin produced mainly by fungi belonging to the genus Fusarium in foods and feeds. It is frequently implicated in reproductive disorders of farm animals and occasionally in hyperoestrogenic syndromes in humans. There is evidence that ZEA and its metabolites possess oestrogenic activity in pigs, cattle and sheep. However, ZEA is of a relatively low acute toxicity after oral or interperitoneal administration in mice, rat and pig. The biotransformation for ZEA in animals involves the formation of two metabolites alpha-zearalenol (alpha-ZEA) and beta-zearalenol (beta-ZEA) which are subsequently conjugated with glucuronic acid. Moreover, ZEA has also been shown to be hepatotoxic, haematotoxic, immunotoxic and genotoxic. The exact mechanism of ZEA toxicity is not completely established. This paper gives an overview about the acute, subacute and chronic toxicity, reproductive and developmental toxicity, carcinogenicity, genotoxicity and immunotoxicity of ZEA and its metabolites. ZEA is commonly found on several foods and feeds in the temperate regions of Europe, Africa, Asia, America and Oceania. Recent data about the worldwide contamination of foods and feeds by ZEA are considered in this review. Due to economic losses engendered by ZEA and its impact on human and animal health, several strategies for detoxifying contaminated foods and feeds have been described in the literature including physical, chemical and biological process. Dietary intakes of ZEA were reported from few countries from the world. The mean dietary intakes for ZEA have been estimated at 20 ng/kgb.w./day for Canada, Denmark and Norway and at 30 ng/kgb.w./day for the USA. The Joint FAO/WHO Expert Committee on Food Additives (JECFA) established a provisional maximum tolerable daily intake (PMTDI) for ZEA of 0.5 microg/kg of body weight [1].

Absorption, Distribution and Excretion
When Leyhorn laying hens were administered (14)C-labeled zearalenone via gavage, 94% of the administered (14)C was excreted in feces within 72 hours. No major retention sites for (14)C activity were identified, but persistent lipophilic metabolites were detected in the yolk. When radiocarbon-labeled zearalenone was administered orally to rats, 70-80% of the recovered fraction was found in feces, and 20-30% in urine. Crystalline zearalenone was added to mixed feed and fed to dairy cows and ewes. Analysis of milk extracts showed trace amounts of zearalenone and β-zearalenol in portions of both milk and sheep milk extracts. A single exposure of laying hens to low concentrations of zearalenone-contaminated feed may not pose a significant health risk to humans. However, long-term exposure could lead to a substantial accumulation of metabolites in the yolk.

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Metabolism/Metabolites
When (14)C-labeled zearalenone was administered by gavage to White Leghorn laying hens, approximately 94% of the (14)C was excreted in feces within 72 hours. One-third of the dose was excreted as unchanged zearalenone, and another third as polar metabolites.
A single administration of 726 mg of zearalenone to pigs was followed by 96 hours of urine collection; 7% of the zearalenone was recovered from the urine after administration, of which 40% was in the form of zearalenol.
The nonsteroidal estrogen zearalenone is metabolized to α-zearalenol in liver homogenate at pH 4.5 and to α-zearalenol and β-zearalenol at pH 7.4.
In the liver of female rats, zearalenone is reduced to zearalenol by 3α-hydroxysterol dehydrogenase.

Zearalenone has been tested for genotoxicity in various experimental systems; all results were negative, with chromosomal aberrations observed only after exposure to very high concentrations in mammalian cells in vitro. Hepatocellular adenomas and pituitary tumors were observed in mouse carcinogenicity studies, but only at doses significantly above the hormonal effect concentration (i.e., ≥8–9 mg/kg bw/d). The committee concluded that these tumors were caused by the estrogenic effect of zearalenone and that the safety of zearalenone could be assessed based on doses that do not produce a hormonal effect in the most sensitive animal—pigs. Based on the no-observed-effect level (NOEL) of 40 µg/kg bw/d in a 15-day study in pigs, the committee determined the permissible tolerable daily intake (PMTDI) of zearalenone to be 0.5 µg/kg bw/d using a safety factor of approximately 100. The Committee also considered the lowest observed adverse event level (LOEL) of 200 µg/kg body weight/day in this study and the previously established acceptable daily intake (ADI) of 0–0.5 µg/kg body weight for the metabolite α-zearalenone (assessed as a veterinary drug). The Committee recommends that the total intake of zearalenone and its metabolites (including α-zearalenone) should not exceed this value.

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Carcinogenicity Evidence
Assessment: There is currently insufficient evidence to suggest that toxins derived from Fusarium graminearum are carcinogenic to humans. There are no data on the carcinogenicity of toxins derived from F. crookwellense and F. culmorum in humans. Limited evidence exists regarding the carcinogenicity of zearalenone in laboratory animals. …Overall Assessment: Toxins derived from Fusarium graminearum, F. culmorum, and F. crookwellense cannot be classified according to their carcinogenicity in humans (Group 3).

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Adverse Reactions
Skin Toxins—Skin burns.
Toxic Pneumonia—Lung inflammation caused by inhalation of metallic fumes or toxic gases and vapors.

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5281576 Rat Oral LD50 >16 gm/kg, Toxicology and Applied Pharmacology, 37(144), 1976; 5281576 Mouse Oral LD50 >2 gm/kg, National Toxicology Program Technical Report Series, NTP-TR-235(1982); 5281576 Mouse Intraperitoneal LD50 5 mg/kg, Veterinary and Human Toxicology, 25(335), 1983 [PMID:6636506]; 5281576 Livestock (Goats/Sheep) Oral LD50 >5 mg/kg, Veterinary and Human Toxicology, 25(335), 1983 [PMID:6636506]

[1]. Review on the toxicity, occurrence, metabolism, detoxification, regulations and intake of zearalenone: an oestrogenic mycotoxin. Food Chem Toxicol. 2007 Jan;45(1):1-18.

[2]. Some major mycotoxins and their mycotoxicoses--an overview. Int J Food Microbiol. 2007 Oct 20;119(1-2):3-10.

Zearalenone is a white microcrystalline or white powder. (NTP, 1992)
Zearalenone is a macrocyclic lactone composed of a fourteen-membered lactone fused with 1,3-dihydroxybenzene; it is a potent estrogenic metabolite produced by certain Fusarium fungi. It is both a fungal metabolite and a fungal estrogen. It is a macrocyclic lactone belonging to the resorcinol class of compounds.
Zearalenone has been reported to exist in Fusarium graminearum, Fusarium equisetifolium, and other organisms with relevant data.
(S-(E))-3,4,5,6,8,10-hexahydro-14,16-dihydroxy-3-methyl-1H-2-benzoxyhexacyclic tetradecene-1,7(8H)-dione. It belongs to the resorcinol acid lactone class of compounds. Cis, trans, dextrorotatory, and levorotatory forms of estrogen have been isolated from Gibberella zeae (formerly Fusarium graminearum). These estrogenic forms are toxic to livestock as feed contaminants and have been used as anabolic agents or estrogen substitutes.

C18H22O5

318.3643

318.146

C, 67.91; H, 6.97; O, 25.13

17924-92-4

5281576

White to off-white solid powder

1.2±0.1 g/cm3

600.4±55.0 °C at 760 mmHg

164-165°C

219.5±25.0 °C

0.0±1.8 mmHg at 25°C

1.539

Fusarium graminearum; Fusarium equiseti; Fusarium

3.83

2

5

0

23

445

1

C[C@H]1CCCC(=O)CCC/C=C/C2=C(C(=CC(=C2)O)O)C(=O)O1

MBMQEIFVQACCCH-QBODLPLBSA-N

InChI=1S/C18H22O5/c1-12-6-5-9-14(19)8-4-2-3-7-13-10-15(20)11-16(21)17(13)18(22)23-12/h3,7,10-12,20-21H,2,4-6,8-9H2,1H3/b7-3+/t12-/m0/s1

(3S,11E)-14,16-dihydroxy-3-methyl-3,4,5,6,9,10-hexahydro-1H-2-benzoxacyclotetradecine-1,7(8H)-dione

ZEA; RAL; F-2 toxin; F 2 toxin; ZEARALENONE; 17924-92-4; (-)-Zearalenone; trans-Zearalenone; Zenone; (S)-Zearalenone; F2 toxin; Mycotoxin F2; Toxin F2

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~314.11 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)